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Correspondencesferrari@imcr.uzh.ch

DisciplinesBiochemistryCancer BiologyCell BiologyMolecular Biology

KeywordsEXO1KAP1DNA DamageMass Spectrometry

Type of ObservationStandalone

Type of LinkStandard Data

Submitted Jun 17th, 2016 Published Aug 29th, 2016

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Triple Blind Peer ReviewThe handling editor, the review-ers, and the authors are all blindedduring the review process.

Full Open AccessSupported by the Velux Foun-dation, theUniversity of Zurich,and the EPFL School of LifeSciences.

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Exonuclease-1 interactswith the transcriptionalco-repressor KAP1MahmoudEl-Shemerly,WassimEid, DanielHess, SerenaBologna, ChristianGentili, StefanoFerrariBasilea Pharmaceutica Ltd.; University of Fribourg, School of Medicine, University of Alexandria, Medical Research Institute(LK); FriedrichMiescher Institute; University of Cambridge, Gurdon Institute; University of Zurich, Institute of Molecular CancerResearch

Error-free repair of DNA double-strand break is orchestrated by homologous recombinationpathways and requires the concerted action of several factors. Among these, EXO1 andDNA2/BLM are responsible for extensive resection of DNA ends to produce 3’-overhangs thatare key intermediates for downstream steps of Homologous Directed Repair (HDR). To helpshed light on regulatory aspects of DNA repair and DNA remodeling pathways in which EXO1participates, and to help reveal novel cellular processes in which EXO1 is involved, we set outto identify proteins interacting with EXO1. Combined immunoprecipitation and mass spec-trometry led to the identification of the non-sense RNA mediated decay protein UPF1 and theDNA damage response protein KAP1/TIF1-β/ TRIM28/RNF96, among others, as EXO1 partners.Follow-up cellular and biochemical studies with recombinant proteins allowed validation of thedirect interaction between EXO1 and KAP1.As a whole, these results provide the basis for future in-depth studies on novel mechanismscontrolling EXO1 and affecting genome stability.

ObjectiveConsidering the multifaceted role of EXO1 in various DNA repair processes, the identi-fication of factors cooperating with EXO1 at sites of damage is expected to shed furtherlight on the function of this important nuclease.

IntroductionThe human genome is continuously challenged by different types of insults, with thou-sands of lesions occurring to DNA every day in each cell (LINDAHL 2000[1]). DNA dam-age either arises as a byproduct of normal cellular metabolism and DNA replication oris induced by external factors (Ciccia 2010[2]). DNA double-strand breaks (DSBs) arethe most dangerous lesions, generated by ionizing radiation (IR), certain chemotherapeu-tic drugs, collapse of stalled DNA replication forks, or caused by endogenous metabolicprocesses, such as meiotic recombination or immunoglobulin diversity generation (Bass-ing 2002[3]) (Whitby 2005[4]) (Errico 2012[5]) (Wyman 2006[6]). DSBs are estimated tooccur at a rate of ten per cell per day in primary human or mouse fibroblasts (Lieber2010[7]). Inappropriate repair of DSBs interferes with DNA replication and transcrip-tion and may cause gross chromosomal aberrations (Eid 2010[8]), resulting in develop-mental defects, neurodegeneration, aging, immunodeficiency, radiosensitivity and steril-ity (Jackson 2009[9]), or facilitating the development of cancer through activation of onco-genes or inactivation of tumor suppressor genes (Curtin 2012[10]). DNA damage response(DDR) pathways have evolved as surveillance and protection mechanisms to counteractthe adverse consequences of DNA lesions and to prevent their transmission to daugh-ter cells (Hoeijmakers 2001[11]). Exonuclease-1 was originally identified in S. pombe(Exo1) ([12]), and subsequently in humans (EXO1) ([13]), as an enzyme belonging tothe Rad2 family of DNA repair nucleases, able to remove mononucleotides from the 5’end of the DNA duplex (Lee 1999[14]). EXO1 is implicated in several DNA repair path-ways, including mismatch repair, post-replication repair, meiotic and mitotic recombina-tion and double-strand break repair (Szankasi 1995[15]) (Fiorentini 1997[16]) ([17]) (Tsub-ouchi 2000[18]) (Mimitou 2009[19]). Human and yeast EXO1 are tightly regulated by in-teraction with CtIP/RBBP8 and 14-3-3 proteins, at DSBs and stalled forks, respectively(Eid 2010[8]) (Engels 2011[20]) (Andersen 2012[21]). Additionally, human EXO1 is con-trolled by post-translational modifications (PTMs), with Ataxia-Telangiectasia and Rad3-related (ATR)-dependent phosphorylation targeting it to ubiquitin-mediated degrada-tion upon replication fork stalling (El Shemerly_2005[22]) (El Shemerly_2007[23]), Ataxia-Telangiectasia-Mutated (ATM)-dependent phosphorylation restraining its activity duringhomologous recombination

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or at uncapped telomeres (Bolderson 2009[24]) (Morin 2008[25]), and CDK-dependent phosphorylation affecting thepathway choice of DSB repair (Tomimatsu 2014[26]). Adding a further layer of complexity, a recent report demonstratedthat EXO1 resection activity is controlled by sumoylation (Bologna 2015[27]). In this study, we report the identificationof novel EXO1 interaction partners and biochemically characterize the interaction with KAP1/TIF1-β/TRIM28/RNF96(hereafter KAP1).

Figure 1 - EXO1 interacts with KAP1(A) Endogenous EXO1 was immunoprecipitated from HEK293T whole cell extract (WCE, 20mg) and proteins were re-solved on an 8% SDS-PAGE gel. Silver stained bands were excised and analyzed by tandem LC-MS/MS. Lane 1: molecularweight markers; lane 2: untreated cells immunoprecipitated with antibody F15; lane 3: untreated cells immunoprecip-itated with pre-immune (PI) serum; lane 4: cells treated with HU (2mM, 16h) and immunoprecipitated with antibodyF15.(B)OMNI-EXO1 and FLAG-KAP1were co-expressed in HEK293T cells. KAP1was immunoprecipitated fromWCE (2mg)using an antibody to the FLAG and proteins were revealed with monoclonal antibodies to EXO1 and the FLAG.(C) FLAG-KAP1 was expressed in HEK293T cells that were either left untreated or treated with HU (2mM, 16h). MG-132(10mM) was added for the same period of time to rescue degradation of EXO1. WCE (5mg) were precipitated with eitherpre-immune serum (PI) or rabbit polyclonal antibody F15 (IP: EXO1). Proteins were revealed with monoclonal antibod-ies to EXO1 and the FLAG tag.(D) Far-Western blot analysis. Upper panel: purified recombinant EXO1 (500ng) and MutS-α (MSH2/MSH6, 500ng)were resolved by SDS-PAGE, transferred to PVDF and stained with Ponceau-red (PR). Lower panels: the membranewas blocked in 5% milk, incubated in the presence or the absence of purified recombinant FLAG-KAP1 and probed withmonoclonal antibodies to the FLAG (center), EXO1 (left) or MSH2/MSH6 (right).(E) HEK293T cells expressing OMNI-EXO1 were either left untreated or treated with HU and MG-132. OMNI-EXO1was immunoprecipitated fromWCE (2mg) using an antibody to the OMNI tag. Proteins were revealed with monoclonalantibodies to MDM2 and EXO1.(F) HEK293T cells were either left untreated or treated with HU and MG-132. Endogenous EXO1 was immunoprecip-itated from WCEs (5mg) with antibody F15 (IP: EXO1). Pre-immune serum (PI) was used as control. Proteins wererevealed with monoclonal antibodies to MDM2 and EXO1.(G)OMNI-EXO1 and HA-MDMXwere co-expressed in HEK293T cells. EXO1was immunoprecipitated fromWCE (2mg)using an antibody to the OMNI tag and proteins were revealed with monoclonal antibodies to the HA-tag and EXO1.(H) HA-MDMX was expressed in HEK293T cells that were either left untreated or treated with HU and MG-132. WCE

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were precipitated with either pre-immune serum (PI) or rabbit polyclonal antibody F15 (IP: EXO1). Proteins were re-vealed with monoclonal antibodies to the HA-tag and EXO1.(I) Far-Western blot analysis. Top panels: 500ng of purified recombinant MutS-α (MSH2/MSH6), KAP1 or EXO1were re-solved by SDS-PAGE, transferred to PVDF and incubated in the presence or the absence of purified recombinant MDM2as indicated. Bottom panels: 500ng of purified recombinant EXO1 or MutS-α (MSH2/MSH6) was resolved as describedabove and incubated in the presence or the absence of purified recombinant MDM2+KAP1 or KAP1 alone, as indicated.All membranes were revealed with a monoclonal antibody to MDM2.(J) Whole cell extracts from HEK-293T cells stably transfected with empty vector (shEV) or KAP1 (shKAP1) shRNAconstructs (left panels) were immunoprecipitated with either rabbit pre-immune serum (PI) or an antibody to EXO1(right panels) and proteins were revealed with the indicated antibodies.(K) Whole cell extracts from HEK-293T cells stably transfected with empty vector (shEV), KAP1 (shKAP1) or MDM2(shMDM2) shRNA constructs and treated in the presence or the absence of HU (2mM, 16h) were immunoprecipitatedas described above and EXO1 was revealed with a mouse monoclonal antibody.(L) Schematic model of the interaction between EXO1 and the KAP1/MDM2/MDMX complex.

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Results and discussionIdentification of EXO1 interacting proteinsExponentially growing HEK-293T cells were either left untreated or treated for 16h with 2mM hydroxyurea (HU) (ElShemerly_2005[22]) (El Shemerly_2007[23]) (Bologna 2015[27]), an inhibitor of the enzyme ribonucleotide reductase(RNR) causing depletion of the pool of dNTPs and early S-phase arrest (Koc 2003[28]). Whole cell extracts (WCE, 20mg)were immunoprecipitated with a rabbit polyclonal antibody to EXO1 and resolved on an 8% SDS-polyacrylamide gel(Fig. 1A). Upon silver staining, the major bands were excised from the gel, submitted to proteolytic digestion and ana-lyzed on a 4000 Q-Trap mass spectrometer (Hess 2008[29]). The data show that, in addition to EXO1, unique peptidesfor established (MSH2) ([30]) and novel partners could be specifically identified (Fig. 1A and Table S1). Genuine novelEXO1 interactors consisted of the GTPase-activating protein TBC1D4, the ATP-dependent helicase RENT1(an essentialfactor of the nonsense-mediated decay (NMD) pathway deputed to the degradation of mRNAs containing prematurestop codons), the leucine zipper protein 1 LUZP1, KAP1, a co-repressor of transcription and SUMO E3-ligase with estab-lished roles in the DNA damage response, the AMP deaminase AMPD2, and the polyadenylate-binding protein 1 PABP1that regulates translational initiation (White 2006[31]) (Li 2007[32]) (Ivanov 2007[33]) (Noon 2010[34]). Interacting pro-teins commonly identified in mass spectrometry studies, such as NCL and HNRNPM, and listed at the ContaminantRepository for Affinity Purification site (www.crapome.org) were not considered further.Analysis of the EXO1-KAP1 interactionTo validate the findings of mass spectrometry studies using independent methods, we focused on KAP1. Ectopic expres-sion of OMNI-EXO1 and FLAG-KAP1 inHEK293T cells followed by immunoprecipitationwith an anti-FLAGmonoclonalantibody confirmed the interaction between the two proteins (Fig. 1B). Immunoprecipitation of the low abundanceEXO1 protein (Eid 2010[8]) (El Shemerly_2005[22]) (El Shemerly_2007[23]) from untreated cells or cells undergoingreplication stress upon treatment with hydroxyurea (HU), revealed constitutive interaction with ectopically expressedFLAG-KAP1 (Fig. 1C). To assess whether the observed protein-protein interaction is direct or is mediated by unknownbridging proteins, we performed Far-Western blot analysis. To this end, purified recombinant EXO1 or the MutSα com-plex (MSH2/MSH6) – used as control – were resolved by SDS-PAGE and transferred to PVDF. Upon visualization of theproteins by Ponceau-red (Fig. 1D, upper panel), the membrane was overlaid with purified recombinant FLAG-KAP1 andsubsequently probed with a monoclonal antibody to the FLAG or control antibodies to EXO1, MSH6 and MSH2. Thedata showed a specific signal for the FLAG in the EXO1 lane only (Fig. 1D, lower panels), confirming that the observedinteraction between EXO1 and KAP1 is direct. Since KAP1 was reported to bind MDM2 and contribute to the functionalregulation of p53 (Wang 2005[35]), we decided to examine whether MDM2 is also part of the EXO1-KAP1 complex. Tothis end, we immunoprecipitated OMNI-EXO1 from extracts of transiently transfected HEK-293T cells. The data showedthat endogenous MDM2 could be found as a constitutive partner of ectopically expressed EXO1 (Fig. 1E). Immunopre-cipitation of endogenous EXO1 confirmed interaction with endogenous MDM2 (Fig. 1F), strengthening the validity ofthis observation. The RING-domain MDM2 ubiquitin E3-ligase interacts with MDMX, which contains a non-functionalRING-domain (Linares 2003[36]). To examine whether MDMX was also part of the protein complex, we ectopically ex-pressed OMNI-EXO1 and HA-MDMX in HEK293T cells. Immunoprecipitation of OMNI-EXO1 from cells treated in thepresence or the absence of HU showed constitutive interaction with MDMX (Fig. 1G). More importantly, we confirmedthat endogenous EXO1 was able to interact with HA-MDMX (Fig. 1H). Finally, we performed Far-Western blot analysiswith purified, recombinant proteins to explore molecular interactions. The data showed that MDM2 directly interactedwith KAP1 but not with EXO1 (Fig. 1I, top panels, lanes 2 and 3) and that the MDM2-EXO1 interaction was only de-tectable upon bridging by KAP1 (Fig. 1I, bottom panels, lane 1). Co-immunoprecipitation experiments confirmed thatin cells where KAP1 expression was lowered by RNA interference, interaction between EXO1 and MDM2 proteins wasdecreased (Fig. 1J).To address the functional role of the EXO1-KAP1-MDM2 interaction, we attenuated expression of either KAP1 orMDM2with specific shRNAs. The data showed that KAP1 depletion did not rescue EXO1 (Fig. 1K, top right panel, lane 2 vs.4), the protein level of which is controlled by ubiquitylation-mediated degradation in response to HU (El Shemerly_-2005[22]). Similar results were obtained upon MDM2 depletion (Fig. 1K, bottom right panel, lane 2 vs. 4), suggestingthat neither KAP1 nor MDM2 is involved in the control of EXO1 protein level.Dysfunction of the machinery that signals DNA damage and/or addresses DNA repair is associated with cancer de-velopment and resistance to therapy (Curtin 2012[10]) (Smits 2014[37]), providing a direct demonstration of the linkbetween genome instability and cancer (Hanahan 2011[38]). Intense effort is currently being devoted to the identi-fication of protein complexes addressing recognition and repair of various forms of DNA damage, as well as to theelucidation of pathways transducing signals to the cell cycle machinery (Bologna 2013[39]) (Reinhardt 2013[40]). Thisknowledge, in turn, is expected to help the development of more efficient drugs that addresses the lack of specificity andside-effects of current chemotherapeutics. The study presented here, focused on an essential component of error-freeDNA repair pathways, contributes to filling this gap through the identification of novel proteins interacting with EXO1,hence expanding our current knowledge (Engels 2011[20]) (Eid 2010[8]). Our data show that EXO1 is part of a multi-protein complex comprising the co-repressor of transcription and E3 SUMO ligase KAP1 and the ubiquitin E3-ligase

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MDM2/MDMX (Fig. 1A-1J and 1L). Interestingly, KAP1 was reported to cooperate with MDM2 in promoting p53 inacti-vation (Wang 2005[35]). However, functional studies that we conducted in cells depleted for KAP1 or MDM2 expressionby RNA interference demonstrated that none of them contributes tomodulating EXO1 protein level in response to stalledDNA replication (Fig. 1K). Hence, the fact that EXO1 is degraded in an ubiquitin- dependent manner upon stalled DNAreplication (Eid 2010[8]) (El Shemerly_2005[22]) (El Shemerly_2007[23]) (Bologna 2015[27]), but that KAP1 or MDM2have no role in this process, indicates that other ubiquitin E3-ligases control EXO1 protein level and, in turn, the extentof DNA resection at sites of damage. A recent study reporting the ability of SCF-Cyclin F to control EXO1 stabilityupon UV-damage, but not in response to ionizing radiation (Elia 2015[41]), indicates that distinct ubiquitin-dependentpathways may be operative in different settings.The low abundance of EXO1, both in yeast (estimated to ~800 molecules per cell in yeast, http://www.yeastgenome.org/)and in humans (El Shemerly_2005[22]) (El Shemerly_2007[23]) (www.proteinatlas.org/), compared to the high expres-sion of KAP1 (www.proteinatlas.org/), suggests that only a subpopulation of KAP1 will be engaged in a stoichiometricinteractionwith EXO1. While KAP1 has also important roles in transcription (Moosmann 1996[42]) (Friedman 1996[43]),the subpopulation of KAP1moleculesmodulating chromatin relaxation (Ryan 1999[44]) in response to DNAdamage (Ziv2006[45]), a function that is under the strict control of PTMs (Li 2007[46]), will likely be engaged in a stoichiometricinteraction with EXO1. We speculate that constitutive physical interaction with a chromatin remodeling factor is ben-eficial to the cell, as it ensures the presence of EXO1 in the vicinity of regions where damage may occur and where,under the control of CtIP (Eid 2010[8]) and PTMs such as phosphorylation, ubiquitylation and sumoylation (El She-merly_2005[22]) (El Shemerly_2007[23]) (Tomimatsu 2014[26]) (Bologna 2015[27]), it can be promptly engaged in therepair of DNA in an error-free manner.

ConclusionsThe identification of novel EXO1 interacting proteins presented in this study, such as mRNA processing factors, DNAbinding proteins, and components of chromatin remodeling factors, represents important additional information onthe complexity of cellular responses to genotoxic damage and offers an opportunity for further in-depth studies on theregulation of the DNA damage response.

Additional Information

Methods and supplementary materialPlease see https://sciencematters.io/articles/201606000006.

Funding statementThis work was supported by grants of Swiss National Science Foundation (SNSF) (31003A_127054; PDFMP3_127523;31003A_144009), Promedica-Stiftung, Hartmann-Müller-Stiftung, Swiss Foundation for Fight Against Cancer, Hermann-Stiftung, Huggenberger-Bischoff-Stiftung, Stiftung für wissenschaftliche Forschung and University of Zurich ResearchFunds.

AcknowledgmentsWe are indebted to Christiane König for invaluable technical support as well as to Aswin Pyakurel and Stefanie Flütschfor help with the characterization of tools and reagents used in this study. We would like to thank members of thelaboratory for critical reading of the manuscript and helpful suggestions.

Ethics statementNot applicable.

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